Project Summary The expected overall impact of this project is to identify developmental and genetic mechanisms underlying tooth formation and replacement. Teeth have classically been used as a model to study organogenesis, as teeth, like most organs, develop through reciprocal epithelial-mesenchymal interactions. Furthermore, 30 percent of people worldwide over the age of 65 have no natural teeth. Thus, knowledge of the developmental and genetic basis of tooth formation and replacement has relevance both for understanding organogenesis, as well as for understanding how teeth can be regenerated in vitro and ultimately in vivo. Although genetic studies in mice and humans have identified signaling pathways involved in tooth development, less is known about genetic mechanisms regulating tooth replacement. Fish replace their teeth constantly throughout adult life, and offer powerful systems for genetic analysis. Here, natural variation in tooth number in the threespine stickleback fish (Gasterosteus aculeatus) is leveraged as a new model system to learn how genes regulate tooth number and tooth replacement. Different stickleback populations adapted to different diets exhibit dramatic heritable changes in tooth number. Two different, independently derived freshwater populations have evolved major increases in tooth number compared to ancestral marine fish. In both high-toothed populations, the tooth number increase arises late in development through an accelerated tooth replacement rate. The different forms can be crossed in the lab, enabling detailed forward genetic analyses to map factors controlling the changes in tooth number. New genome editing methods allow functional tests of genes and cis-regulatory elements of interest. A cis-regulatory allele of the Bone Morphogenetic Protein 6 (Bmp6) gene is associated with evolved tooth gain in one high-toothed population. Pharmacological and genetic data suggest BMP signaling and Bmp6 positively regulate primary tooth number, but inhibit tooth replacement. To test hypotheses about the developmental and genetic bases of tooth formation and replacement, three specific aims are proposed. First, we will test whether BMP signaling and Bmp6 regulate dental stem cell quiescence during tooth replacement by BrdU and vital dye pulse-chase labeling, gene expression, and pharmacological experiments. Second, we will identify upstream regulators of two Bmp6 enhancers and determine enhancer and regulator functions during tooth development and replacement by pharmacological, transgenic, and genome editing experiments. Third, we will identify the genetic basis of evolved tooth gain in an independently derived high-toothed freshwater population with a distinct developmental genetic basis by a combination of genetic mapping, genome editing, and gene expression experiments. Together these results will shed new light on developmental and genetic mechanisms underlying tooth replacement.